Project Summary/Abstract Mammalian nuclei are subdivided by a host of membrane-less organelles (MLOs) that separate from the surrounding nucleoplasm to serve distinct functions. Nuclear speckles are among the most prominent MLOs in mammalian nuclei, with 20-50 speckles per cell, each measuring ~1 m in diameter. Speckles contain hundreds of RNA binding proteins (RBPs) involved in several mRNP maturation events ? including pre-mRNA splicing and mRNP nuclear export ? and also contain the highest concentration of post-transcriptional RNA in the nucleus. Many components of speckles are oncogenes, and drugs that modulate speckles are anti-tumorigenic. Yet despite their discovery over 50 years ago, the molecular function of speckles is unknown. The RNA within speckles has not been defined, nor is it clear how speckles affect RNA processing or mRNP assembly. This proposal employs novel strategies to elucidate the composition, regulation, and function of speckles, and will directly test the hypothesis that speckles impact mRNP maturation and alter mammalian gene expression. The premise of this work is that investigating the function of speckles requires the study of entire speckles, and not just the study of individual factors within them. Aim 1 will result in the development of a robust speckle- purification method that uses fluorescent-particle sorting to isolate GFP-labeled speckles from cell lysate. Quantitative proteomics and RNA-sequencing of purified speckles will provide the first comprehensive analysis of their composition. Subsequent analysis will determine whether all or a subset of mammalian mRNPs traffic through speckles, and will define whether RNAs are detained in speckles due to specific sequences and/or incomplete splicing. Aim 2 will assess how speckles directly impact mRNP maturation. Two kinases that naturally promote speckle disassembly in distinct physiological contexts will be separately hyperactivated or inhibited, followed by genome-wide analysis of pre-mRNA splicing and nuclear export. Identifying common effects between two kinases will reveal how speckle disassembly impacts mRNP maturation, as opposed to other effects of each individual kinase. Aim 3 will define the RNA-protein interactions within speckles for essential RBPs. UV- crosslinking followed by speckle-purification will identify speckle-specific RNA interactions for two SR proteins, a family of RBPs that promote splicing and export of thousands of mRNAs. This analysis will reveal whether RBPs within speckles bind to substrate RNAs that are unspliced, alternatively spliced, or consitutively spliced isoforms. Ultimately, the methods and results stemming from this work will serve as a platform to investigate speckles in the context of development and disease. The proposed research goals will provide significant training in the use of many genome-wide approaches, including RNA-seq, proteomics, and eCLIP, as well as training in modern genetic manipulations of human cells. The research will take place in the lab of Dr. Karla Neugebauer, an outstanding RNA biologist at Yale University who provides both professional and scientific mentorship.